Nanocrystals having polynucleotide strands and their use to form dendrimers in a signal amplification system

ABSTRACT

Provided are compositions and assay kits comprising functionalized nanocrystals having extending therefrom a plurality of polynucleotide strands of known sequence; wherein primary dots are used to operably link to a molecular probe, and secondary dots comprise a plurality of polynucleotide strands which are complementary to the plurality of polynucleotide strands of the primary dots. Also provided is a method for detecting the presence or absence of target molecule in a sample comprising operably linking primary dots to molecular probe, contacting the complex formed with the sample, contacting the sample with successive additions of secondary dots and primary dots. If target molecule is present in the sample, the primary dots and secondary dots will form dendrimers that can be detected by fluorescence emission.

RELATED APPLICATIONS

This is a continuation-in-part patent application based on earlier,co-pending patent application Ser. No. 09/437,076 which is hereinincorporated by reference.

FIELD OF INVENTION

This invention relates to novel compositions comprising functionalizednanocrystals. More particularly, the present invention relates towater-soluble nanocrystals which further comprise strands ofpolynucleotides of a known sequence, and the use of such functionalizednanocrystals to provide signal and signal amplification for detectingtarget molecules.

BACKGROUND OF THE INVENTION

Nonisotopic detection systems have become a preferred mode in scientificresearch and clinical diagnostics for the detection of biomoleculesusing various assays including, but not limited to, flow cytometry,nucleic acid hybridization, DNA sequencing, nucleic acid amplification,immunoassays, histochemistry, and functional assays involving livingcells. In particular, while fluorescent organic molecules such asfluorescein and phycoerythrin are used frequently in detection systems,there are disadvantages in using these molecules in combination. Forexample, each type of fluorescent molecule typically requires excitationwith photons of a different wavelength as compared to that required foranother type of fluorescent molecule. However, even when a single lightsource is used to provide a single excitation wavelength (in view of thespectral line width), often there is insufficient spectral spacingbetween the emission optima of different fluorescent molecules to permitindividual and quantitative detection without substantial spectraloverlap. Additionally, conventional fluorescent have limitedfluorescence intensity. Further, currently available nonisotopicdetection systems typically are limited in sensitivity due to the finitenumber of nonisotopic molecules which can be used to label a biomoleculeto be detected.

Branched DNA or DNA dendrimers have been constructed as a signalamplification tool (see, e.g., U.S. Pat. Nos. 5,487,973, 5,484,904, and5,175,270). These matrices are comprised of a DNA backbone having DNAarms. For example, matrices are constructed of successive subunits of adouble-stranded DNA with single stranded arms on each end. Some of thearms are used to hybridize to a specific oligonucleotide probe, whereasother arms are used to bind to a nonisotopic or isotopic label. Whileproviding for the addition of an relative increase in the number oflabel molecules as compared to other systems, one disadvantage is that asubunit needs to be custom-synthesized to contain at least one armconsisting of a complementary sequence which is capable of hybridizingto the specific DNA sequence which the user wishes to detect.Additionally, the label molecules are attached or ligated to theoutwardly extending ends (tips) of the DNA matrices, rather than as anintegral part of the matrix.

Thus, there remains a need for a nonisotopic detection system which (a)can result in generation of a signal comprising fluorescence emission ofhigh quantum yield; (b) can result in signal amplification; (c) is notlimited as to the chemical nature of the target molecule to be detected(e.g., versus detection of nucleic acid molecules only); (d) can be usedto bind molecular probes of various types (versus binding tooligonucleotide probes only); (e) is preferably universal in terms ofdetecting target molecules of various sequences; and (f) can result inthe simultaneous detection of more than one type of target molecule byutilizing a class of nonisotopic molecules that may be excited with asingle excitation light source and with resultant fluorescence emissionswith discrete fluorescence peaks.

SUMMARY OF THE INVENTION

The present invention provides methods, compositions, and kits for usein an amplifiable, non-isotopic detection systems. The compositioncomprises nanocrystals that are functionalized to be water-soluble, andfurther functionalized to comprise a plurality of polynucleotide strandsof a known (predetermined) sequence which extend outwardly from eachnanocrystal. While there are several variations of this system, a basicprinciple of the invention is that a molecular probe is used to detect atarget molecule, if present in a sample, by the binding specificity ofthe molecular probe for the target molecule or a portion thereof; andgeneration and amplification of a detectable signal by using at leasttwo species of functionalized nanocrystals. A first species offunctionalized nanocrystals (“primary dots”) have extending therefromstrands of polynucleotides of known sequence, and wherein the primarydots are, or become, operably linked to the molecular probe. A secondspecies of functionalized nanocrystals (“secondary dots”) also havestrands of polynucleotides of known sequence extending therefrom,wherein the nucleic acid sequence of the polynucleotide strands of thesecondary dots is sufficiently complementary to the nucleic acidsequence of the polynucleotide strands on the primary dots such that,under suitable conditions for promoting contact and hybridization, therespective complementary strands hybridize to each other in forming adendrimer. In multiple steps in which subsequent additions offunctionalized nanocrystals alternate between primary dots and secondarydots, a dendrimer of multiple layers of functionalized nanocrystals isformed, thereby resulting in a detectable signal and an exponentialincrease in the amount of detectable signal that can be detected from asingle molecular probe. Additionally provided, are assay kits comprisingreagents for the signal amplification system according to the presentinvention.

The above and other objects, features, and advantages of the presentinvention will be apparent in the following Detailed Description of theInvention when read in conjunction with the accompanying drawings inwhich reference numerals denote the same or similar parts throughout theseveral illustrated views and embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. is a schematic illustrating functionalizing a water-solublenanocrystal containing a layer of a capping compound to further comprisea coating of a diaminocarboxylic acid, and then operably linking thediaminocarboxylic acid to a molecular probe.

FIG. 2A is a bar graph comparing the stability of capped quantum dots(“W-SN”) to the stability of functionalized nanocrystals (“FN”) underoxidizing conditions.

FIG. 2B is a bar graph comparing the non-specific binding of cappedquantum dots (“W-SN”) to the non-specific binding of functionalizednanocrystals (“FN”).

FIG. 3 is a schematic illustration showing a functionalized here asbound to its target molecule); and showing dendrimer formation resultingfrom subsequent and successive additions of secondary dots and primarydots.

FIG. 4A is a schematic illustration showing an avidinylatedfunctionalized nanocrystal comprising a primary dot operably linked to amolecular probe comprising a biotinylated oligonucleotide (which isshown here as bound to its target molecule); and showing dendrimerformation resulting from subsequent and successive additions ofsecondary dots and primary dots.

FIG. 4B is a schematic illustration showing an avidinylatedfunctionalized nanocrystal comprising a primary dot which is operablylinked to a molecular probe comprising a biotinylated antibody (which isshown here as bound to its target molecule); and showing dendrimerformation resulting from subsequent and successive additions ofsecondary dots and primary dots.

FIG. 5 is a schematic illustration showing an avidin intermediate whichis used to operably link biotinylated polynucleotide strands of afunctionalized nanocrystal comprising a primary dot to a biotinylatedmolecular probe (which is shown here as bound to its target molecule);and showing dendrimer formation resulting from subsequent and successiveadditions of secondary dots and primary dots.

FIG. 6A is a schematic illustration showing use of a linker to operablylink a polynucleotide strand of a functionalized nanocrystal to amolecular probe (step 1), and subsequent interaction of the molecularprobe with its target molecule (step 2).

FIG. 6B is a schematic illustration showing a process (step 3) continuedfrom FIG. 6A, wherein subsequent and successive additions of secondarydots and primary dots result in dendrimer formation.

FIG. 7 is a schematic illustration showing use of a nucleic acidmolecule linker which hybridizes to both a polynucleotide strand of afunctionalized nanocrystal and to a molecular probe comprising anoligoncleotide, in operably linking the primary dot to the molecularprobe; and showing dendrimer formation resulting from subsequent andsuccessive additions of secondary dots and primary dots.

FIG. 8 is a schematic illustration showing a molecular probe which isincorporated or synthesized as part of a polynucleotide strand of afunctionalized nanocrystal comprising a primary dot (wherein themolecular probe portion is shown here as bound to its target molecule);and showing dendrimer formation resulting from subsequent and successiveadditions of secondary dots and primary dots.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Throughout the specification of the application, various terms are usedsuch as “primary”, “secondary”, “first”, “second”, and the like. Theseterms are words of convenience in order to distinguish between differentelements, and such terms are not intended to be limiting as to how thedifferent elements may be utilized.

By the term “target molecule” is meant, for the purposes of thespecification and claims to refer to a molecule of an organic orinorganic nature, the presence and/or quantity of which is being testedfor; and which contains a molecular component (e.g., ligand or sequenceor epitope or domain or portion or chemical group or reactivefunctionality or determinant) for which a molecular probe has bindingspecificity. The molecule may include, but is not limited to, a nucleicacid molecule, protein, glycoprotein, eukaryotic cell, prokaryotic cell,lipoprotein, peptide, carbohydrate, lipid, phospholipid, aminoglycans,chemical messenger, biological receptor, structural component, metabolicproduct, enzyme, antigen, drug, therapeutic, toxin, inorganic chemical,organic chemical, a substrate, and the like. The target molecule may bein vivo, in vitro, in situ, or ex vivo.

By the term “molecular probe” is meant, for purposes of thespecification and claims, to mean a molecule which has bindingspecificity and avidity for a molecular component of, or associatedwith, a target molecule. In general, molecular probes are known to thoseskilled in the art to include, but are not limited to, lectins orfragments (or derivatives) thereof which retain binding function,monoclonal antibodies (“nab”, including chimeric or genetically modifiedmonoclonal antibodies which may be preferable for administration tohumans), peptides, aptamers, and nucleic acid molecules (including, butnot limited to, single stranded RNA or single-stranded DNA, orsingle-stranded nucleic acid hybrids, oligonucleotide analogs, backbonemodified oligonucleotide analogs, morpholino-based polymers). The term“monoclonal antibody” is also used herein, for purposes of thespecification and claims, to include immunoreactive fragments orderivatives derived from a mAb molecule, which fragments or derivativesretain all or a portion of the binding function of the whole mAbmolecule. Such immunoreactive fragments or derivatives are known tothose skilled in the art to include F(ab′)₂, Fab′, Fab, Fv, scFV, Fd′and Fd fragments. Methods for producing the various fragments orderivatives from mAbs are well known in the art (see, e.g., Plückthum,1992, Immunol. Rev. 130:152-188). For example, F(ab′)₂ can be producedby pepsin digestion of the monoclonal antibody, and Fab′ may be producedby reducing the disulfide bridges of F(ab′)₂ fragments. Fab fragmentscan be produced by papain digestion of the monoclonal antibody, whereasFv can be prepared according to methods described in U.S. Pat. No.4,642,334. Single chain antibodies can be produced as described in U.S.Pat. No. 4,946,778. The construction of chimeric antibodies is now astraightforward procedure (Adair, 1992, Immunological Reviews 130:5-40,) in which the chimeric antibody is made by joining the murinevariable region to a human constant region. Additionally, “humanized”antibodies may be made by joining the hypervariable regions of themurine monoclonal antibody to a constant region and portions of variableregion (light chain and heavy chain) sequences of human immunoglobulinsusing one of several techniques known in the art (Adair, 1992, supra;Singer et al., 1993, J. Immunol. 150:2844-2857). Methods for making achimeric non-human/human mAb in general are described in detail in U.S.Pat. No. 5,736,137. Aptamers can be made using methods described in U.S.Pat. No. 5,789,157. Lectins and fragments thereof are commerciallyavailable. Oligonucleotide analogs, backbone modified oligonucleotideanalogs, and morpholino-based polymers can be made using methodsdescribed in U.S. Pat. Nos. 5,969,135, and 5,596,086, U.S. Pat. Nos.5,602,240, and 5,034,506, respectively. “Molecular probe” is often usedherein, particularly when referring to adding the molecular probe to asample for detection purposes, or operably linking to a plurality ofpolynucleotide strands of primary dots, as meaning a plurality ofmolecules of the molecular probe.

By the term “hybridize” is meant, for purposes of the specification andclaims, to refer to a process by which a single-stranded nucleic acidmolecule joins with a complementary strand through nucleotide basepairing. As apparent in the art, a sufficient number of complementarybase pairs are needed for hybridization, and the selectivity ofhybridization depends on the degree of complementarity, the stringencyof conditions during the hybridization process, and the length of thehybridizing strands. In a preferred embodiment, nucleic acid molecules,and more particularly polynucleotide strands, which are complementaryare hybridizable under “suitable conditions”; i.e., under optimalreaction conditions of temperature, ionic strength, and time of reactionwhich permit hybridization between the desired complementary nucleicacid molecules and that minimize nondiscriminate hybridization (e.g.,disfavor non-homologous base pairing). Typically, such conditions aredescribed ranging from medium stringency to high stringency. Likewise,where a polynucleotide strand lacks sufficient complementarity tospecifically hybridize to another polynucleotide strand means that thestrands lack sufficient complementarity to hybridize under conditionsranging form medium stringency to high stringency, and more preferably,lack sufficient complementarity to hybridize under conditions rangingfrom low stringency to high stringency, as understood by those skilledin the art. Also in a preferred embodiment, the two strands which arehybridized are of sufficient complementarity and length that theresulting base paired complex (hybrid) is sufficiently stable to servethe functions of dendrimer formation and detection as described in moredetail herein.

By the term “effective amount” is meant, for purposes of thespecification and claims and when used in conjunction with a molecule orcompound described herein, to refer an amount sufficient to contact andoperably link its target for which it has binding specificity (ifpresent in the mixture) for the purposes of signal amplification anddetection according to the present invention. For example, an effectiveamount of secondary dots means that added to a mixture is a sufficientamount of secondary dots to hybridize with primary dots containingcomplementary polynucleotide strands, if such primary dots are present.In a preferred embodiment, an effective amount comprises an amount thatwould saturate (e.g., bind substantially all available) any specific andavailable binding sites of its target (e.g., if a secondary dot, bind toall available primary dots containing complementary polynucleotidestrands).

By the term “operably linked” is meant, for purposes of thespecification and claims to refer to fusion or bond or an association,of sufficient stability for the purposes of signal amplification anddetection according to the present invention, between a combination ofdifferent molecules such as, but not limited to, between a linker and amolecular probe, between a molecular probe and the terminal portion ofone or more polynucleotide strands of a primary dot; between a linkerand a primary dot; between a molecular probe and a primary dot; betweena terminal portion of a polynucleotide strand and a coat of thenanocrystal portion of a primary dot; between a fluorescent nanocrystaland a capping compound; between a capping compound and adiaminocarboxylic acid; between a diaminocarboxylic acid and adiaminocarboxylic acid; between a diaminocarboxylic acid and a linker;between a diaminocarboxylic acid and an amino acid; between an aminoacid and a molecular probe; between a diaminocarboxylic acid and apolynucleotide strand; and a combination thereof. As known to thoseskilled in the art, and as will be more apparent by the followingembodiments, there are several methods and compositions in which two ormore molecules may be operably linked utilizing reactivefunctionalities. Reactive functionalities include, but are not limitedto, bifunctional reagents/linker molecules, biotin, avidin, freechemical groups (e.g., thiol, or carboxyl, hydroxyl, amino, amine,sulfo, etc.), and reactive chemical groups (reactive with free chemicalgroups).

By the term “linker” is meant, for purposes of the specification andclaims to refer to a compound or moiety that acts as a molecular bridgeto operably link two different molecules, wherein one portion of thelinker is operably linked to a first molecule, and wherein anotherportion of the linker is operably linked to a second molecule. The twodifferent molecules may be linked to the linker in a step-wise manner.There is no particular size or content limitations for the linker solong as it can fulfill its purpose as a molecular bridge. Linkers areknown to those skilled in the art to include, but are not limited to,chemical chains, chemical compounds, carbohydrate chains, peptides,haptens, and the like. The linkers may include, but are not limited to,homobifunctional linkers and hetero-bifunctional linkers.Heterobifunctional linkers, well known to those ski).led in the art,contain one end having a first reactive functionality to specificallylink a first molecule, and an opposite end having a second reactivefunctionality to specifically link to a second molecule. As illustrativeexamples, to operably link a hydroxyl group of a polynucleotide strandof a primary dot to the amino group of a molecular probe, the linker mayhave: a carboxyl group to form a bond with the polynucleotide strand,and a carboxyl group to form a bond with the molecular probe; or acarboxyl group to form a bond with the polynucleotide strand, and analdehyde group to form a bond with the molecular probe; or a carboxylgroup to form a bond with the polynucleotide strand, and a halide groupto form a bond with the molecular probe. In another example, to operablylink a carboxyl group of a polynucleotide strand of a primary dot to theamino group of a molecular probe, the linker can have: an amino group toform a bond with the polynucleotide strand, and a carboxyl group to forma bond with the molecular probe; or an amino group to form a bond withthe polynucleotide strand, and a hydroxyl group to form a bond with themolecular probe; or a hydroxyl group to form a bond with thepolynucleotide strand, and a sulphonic acid group to form a bond withthe molecular probe (see, e.g., U.S. Pat. Nos. 5,792,786, and 5,780,606for various linkers known in the art). A linker may comprise a primaryamine reactive group to react with an amino acid (e.g., lysine) residueof a molecular probe comprising a mAb, and a thiol reactive group toreact with a terminally thiolated polynucleotide strand of a primarydot. Heterobifunctional photo-reactive linkers (e.g., phenylazidescontaining a cleavable disulfide bond) are known in the art. Forexample, a sulfo-succinimidyl-2-(p-azido salicylamido)ethyl-1,3′-dithiopropionate contains a N-hydroxy-succinimidyl groupreactive with primary amino groups, and the phenylazide (uponphotolysis) reacts with any amino acids. The linker may further comprisea protective group which blocks reactivity with a functional group onthe linker which is used to react with and bind to a molecule to belinked. A deprotection reaction may involve contacting the linker to oneor more conditions and/or reagents which removes the protective group,thereby exposing the functional group to interact with the molecule tobe linked. Depending on the nature of the protective group, deprotectioncan be achieved by various methods known in the art, including, but notlimited to photolysis, acidolysis, hydrolysis, and the like. Dependingon such factors as the molecules to be linked, and the conditions inwhich the method of detection is performed, the linker may vary inlength and composition for optimizing such properties as flexibility,stability, and resistance to certain chemical and/or temperatureparameters. For example, short linkers of sufficient flexibilityinclude, but are not limited to, linkers having from 2 to 10 carbonatoms (see, e.g., U.S. Pat. No. 5,817,795).

By the term “dendrimer” is meant, for purposes of the specification andclaims to refer to a matrix formed by interaction between two or morespecies of functionalized nanocrystals, wherein the matrix formation isfacilitated by the interaction of polynucleotide strands betweenrespective species of functionalized nanocrystals, and wherein apolynucleotide strand becomes held in close association via base pairingand/or covalent crosslinking with a complementary strand.

By the term “strand” is meant, when used in conjunction with or inreference to the term “polynucleotide” and for purposes of thespecification and claims to refer to a nucleic acid molecule which issubstantially single-stranded (comprising all or a substantial portionof single-strandedness sufficient to hybridize with a complementarysingle strand in the formation of a dendrimer). There is no particularsize, length or content limitations for the strand, so long as it canfulfill its purpose in dendrimer formation as described herein. Thenucleic acid composition of the polynucleotides may be selected frommolecules which include, but are not limited to, ribonucleotides (RNA),deoxyribonucleotides (DNA), RNA/DNA hybrids, naturally occurringnucleotides, and synthetic or modified nucleotides (e.g.,oligonucleotide analogs, backbone modified oligonucleotide analogs,morpholino-based polymers, and the like).

By the term “diaminocarboxylic acid” is meant, for purposes of thespecification and claims to refer to an amino acid that has two freeamine groups. The amino acid may be a naturally occurring amino acid, asynthetic amino acid, a modified amino acid, an amino acid derivative,an amino acid precursor (e.g., citrulline and ornithine areintermediates in the synthesis of arginine), or a combination thereof.In a preferred embodiment, the diaminocarboxylic acid contains neutral(uncharged) polar functional groups which can hydrogen bond with water,thereby making the diaminocarboxylic acid (and the quantum dot to whichit is made a part of) relatively more soluble in aqueous solutionscontaining water than those with nonpolar functional groups.Additionally, the diaminocarboxylic acid imparts one or more functionaladvantages to the water-soluble nanocrystal of which it is a part, aswill be more apparent from the following embodiments. Exemplarydiaminocarboxylic acids include, but are not limited to, lysine,asparagine, glutamine, arginine, citrulline, ornithine, 5-hydroxylysine,djenkolic acid, β-cyanoalanine, a synthetic diaminocarboxylic acid(e.g., such as 3,4-diaminobenzoic acid, 2,3-diaminopropionic acid,2,4-diaminobutyric acid, 2,5-diaminopentanoic acid, 2,6-diaminopimelicacid), and a combination thereof. A diaminocarboxylic acid of theaforementioned diaminocarboxylic acids may be used in a preferredembodiment, and a preferred diaminocarboxylic acid may be usedseparately in the method according to the present invention to theexclusion of diaminocarboxylic acids other than the preferreddiaminocarboxylic acid.

By the term “amino acid” is meant, for purposes of the specification andclaims to refer to a molecule that has at least one free amine group andat least one free carboxyl group. The amino acid may have more than onefree amine group, or more than one free carboxyl group, or may furthercomprise one or more free chemical reactive groups other than an amineor a carboxyl group (e.g., a hydroxyl, a sulfhydryl, etc.). The aminoacid may be a naturally occurring amino acid, a synthetic amino acid, amodified amino acid, an amino acid derivative, and an amino acidprecursor. The amino acid may further be selected from the groupconsisting of a monoaminocarboxylic acid, and a diaminocarboxylic acid.In a preferred embodiment, the monoaminocarboxylic acid contains one ormore neutral (uncharged) polar functional groups which can hydrogen bondwith water, thereby making the monoaminocarboxylic acid (and the quantumdot to which it is made a part of) relatively more soluble in aqueoussolutions containing water than those with non-polar functional groups.Exemplary monoaminocarboxylic acids include, but are not limited to,glycine, serine, threonine, cysteine, β-alanine, homoserine,γ-aminobutyric acid, and a combination thereof. An amino γ-aminobutyricacid, and a combination thereof. An amino acid of the aforementionedamino acids may be used in a preferred embodiment, and a preferred aminoacid may be used separately in the method according to the presentinvention to the exclusion of amino acids other than the preferred aminoacid.

By the term “capping compound” is meant, for purposes of thespecification and claims to refer to a compound selected from the groupconsisting of an amino acid, a compound having the formula HS(CH₂)_(n)X,wherein X is a carboxylate (carboxylic moiety), and a compound havingthe formula HS(CH₂)_(n)YX, wherein X is a carboxylate and Y is an amine,as will be more apparent from the following descriptions. “n” is anumber in the range of from 1 to about 20, and preferably greater than4. A reactive group of the capping compound operably links with thefluorescent nanocrystal creating a layer which is not easily displacedin solution. Additionally, the reactive group or moiety and/or the amineof the capping compound preferably imparts some water solubility to thecapped fluorescent nanocrystals. Exemplary capping compounds include,but are not limited to, mercaptocarboxylic acid, ormercaptofunctionalized amines (e.g., aminoethanethiol-HCl, homocysteine,or 1-amino-2-methyl-2-propanethiol-Hcl), or a combination thereof. Acapping compound of the aforementioned capping compounds may be used ina preferred embodiment, and a preferred capping compound may be usedseparately in the method according to the present invention to theexclusion of capping compounds other than the preferred cappingcompound.

By the term “fluorescent nanocrystals” is meant, for purposes of thespecification and claims to refer to nanocrystals comprisingsemiconductor nanocrystals or doped metal oxide nanocrystals.“Semiconductor nanocrystals” is meant, for purposes of the specificationand claims to refer to quantum dots (crystalline semiconductors)comprised of a core comprised of at least one of a Group II-VIsemiconductor material (of which ZnS, and CdSe are illustrativeexamples), or a Group III-V semiconductor material (of which GaAs is anillustrative example), a Group IV semiconductor material, or acombination thereof. In a preferred embodiment, the core of the quantumdots may be passivated with an semiconductor overlayering (“shell”)uniformly deposited thereon. For example, a Group II-VI semiconductorcore may be passivated with a Group II-VI semiconductor shell (e.g., aZnS or CdSe core may be passivated with a shell comprised of YZ whereinY is Cd or Zn, and Z is S, or Se). As known to those skilled in the art,the size of the semiconductor core correlates with the spectral range ofemission. Table 1 is an illustrative example for CdSe.

TABLE 1 Color Size Range (nm) Peak Emission Range blue  2.5 to 2.68 476to 486 green 2.8 to 3.4 500 to 530 yellow 3.58 to 4.26 536 to 564 orange4.9 to 6.1 590 to 620 red  8.6 to 10.2 644 to 654

In a preferred embodiment, the semiconductor nanocrystals are producedusing a continuous flow process and system disclosed in U.S. Pat. No.6,179,912 (the disclosure of which is herein incorporated by reference),and have a particle size that varies by less than +/−4% in the averageparticle size. In a preferred embodiment, the semiconductor nanocrystalscomprise a monodisperse population having an average particle size (asmeasure by diameter) in the range of approximately 1 nanometer (nm) toapproximately 20 nm. By the term “doped metal oxide nanocrystals” ismeant, for purposes of the specification and claims to refer tonanocrystals comprised of: a metal oxide, and a dopant comprised of oneor more rare earth elements. For example, suitable metal oxides include,but are not limited to, yttrium oxide (Y₂0₃), zirconium oxide (Zr0₂),zinc oxide (ZnO), copper oxide (CuO or Cu₂0), gadolinium oxide (Gd₂0₃),praseodymium oxide (Pr₂0₃), lanthanum oxide (La₂0₃), and alloys thereof. The rare earth element comprises an element selected from theLanthanide series and includes, but is not limited to, europium (Eu),cerium (Ce), neodymium (Nd), samarium (Sm), terbium (Tb), gadolinium(Gd), holmium (Ho), thulium (Tm), an oxide thereof, and a combinationthereof. As known to those skilled in the art, depending on the dopant,an energized doped metal oxide nanocrystal is capable of emitting lightof a particular color. Thus, the nature of the rare earth or rare earthsare selected in consequence to the color sought to be imparted (emitted)by a doped metal oxide nanocrystal used to label a microsphere accordingto the present invention. A given rare earth or rare earth combinationhas a given color, thereby permitting the provision of doped metal oxidenanocrystals, each of which may emit (with a narrow emission peak) acolor over an entire range of colors by adjusting the nature of thedopant, the concentration of the dopant, or a combination thereof. Forexample, the emission color and brightness (e.g., intensity) of a dopedmetal oxide nanocrystal comprising Y₂0₃:Eu may depend on theconcentration of Eu; e.g., emission color may shift from yellow to redwith increasing Eu concentration. For purposes of illustration only,representative colors which may be provided are listed in Table 2.

TABLE 2 Florescent Color Dopant blue Thulium blue Cerium yellow-greenTerbium green Holmium green Erbium red Europium reddish orange Samariumorange Neodymium yellow Dysprosium white Praseodymium orange-yelloweuropium + terbium orange-red europium + samarium

Methods for making doped metal oxide nanocrystals are known to include,but are not limited to a sol-gel process (see, e.g., U.S. Pat. No.5,637,258), and an organometallic reaction. As will be apparent to oneskilled in the art, the dopant (e.g., one or more rare earth elements)are incorporated into the doped metal oxide nanocrystal in a sufficientamount to permit the doped metaloxide nanocrystal to be put to practicaluse in fluorescence detection as described herein in more detail. Aninsufficient amount comprises either too little dopant which would failto emit sufficient detectable fluorescence, or too much dopant whichwould cause reduced fluorescence due to concentration quenching. In apreferred embodiment, the amount of dopant in a doped metal oxidenanocrystal is a molar amount in the doped metal oxide nanocrystalselected in the range of from about 0.1% to about 25%. Doped metal oxidenanocrystals may can be excited with a single excitation light sourceresulting in a detectable fluorescence emission of high quantum yield(e.g., a single quantum dot having at a fluorescence intensity that maybe a log or more greater than that a molecule of a conventionalfluorescent dye) and with a discrete fluorescence peak. Typically, theyhave a substantially uniform size of less than 200 Angstroms, andpreferably have a substantially uniform size in the range of sizes offrom about 1 nm to about 5 nm, or less than 1 nm. In a preferredembodiment, the doped metal oxide nanocrystals are comprised of metaloxides doped with one or more rare earth elements, wherein the dopantcomprising the rare earth element is capable of being excited (e.g.,with ultraviolet light) to produce a narrow spectrum of fluorescenceemission. In another preferred embodiment, the doped metal oxide hasboth fluorescent properties (when excited with an excitation lightsource) and magnetic properties; thus, a polymeric microsphere (which issubstantially nonmagnetic) embedded with a plurality of fluorescentnanocrystals (comprising doped metal oxide nanocrystals which aremagnetic material) may form fluorescent microspheres according to thepresent invention which are magnetic.

By the term “operably link” is meant, for purposes of the specificationand claims to refer to fusion or bond or an association of sufficientstability to withstand conditions encountered in a method of detection,between a combination of different molecules such as, but not limitedto, between a fluorescent nanocrystal and the molecules by which theyare functionalized (e.g., carboxylic acid, diaminocarboxylic acid, or amonoaminocarboxylic acid), a fluorescent microsphere and affinityligand, and a combination thereof. As known to those skilled in the art,the bond may comprise one or more of covalent, ionic, hydrogen, van derWaals, and the like. As known to those skilled in the art, and as willbe more apparent by the following embodiments, there are several methodsand compositions in which two or more molecules may be operably boundutilizing reactive functionalities.

By the term “functionalized nanocrystals” is meant, for purposes of thespecification and claims to refer to fluorescent nanocrystals which arecoated with at least one coating that (a) enhances stability and/orsolubility in an aqueous environment; (b) provides one or more reactivefunctionalities that may be used to operably link the functionalizednanocrystal to a plurality of polynucleotide strands, or to a linker(one or more linker molecules) which may then be used to operably linkthe functionalized nanocrystal to a plurality of polynucleotide strands;and (c) wherein a coating of the at least one coating comprises aminoacid (e.g., the coating comprises a capping compound comprising an aminoacid; or amino acid is an additional coating that coats (is operablylinked to) a capping compound, wherein the capping compound is otherthan amino acid; or amino acid coats a capping compound wherein thecapping compound comprises amino acid). Preferably, a functionalizednanocrystal may further comprise a plurality of polynucleotide strandsoperably linked thereto. In a preferred embodiment, the at least oneadditional coat comprises amino acid, wherein each coating of amino acidcontains neutral (uncharged) polar functional groups which can hydrogenbond with water, and comprises one or more types of free chemicalreactive groups. In a more preferred embodiment, the at least one layerof amino acid that functionalizes the nanocrystal is comprised of acoating of diaminocarboxylic acid. A preferred functionalizednanocrystal may be produced, and used in the method and system accordingto present invention, to the exclusion of functionalized nanocrystalsother than the preferred functionalized nanocrystals

The present invention provides compositions which can be used to buildthree dimensional dendrimers which function to generate andsignificantly amplify a detectable signal, thereby considerablyimproving the sensitivity of a non-isotopic detection system. The use ofpolynucleotide strands to build the dendrimers is fundamental to thisinvention. Also fundamental to the invention are functionalizednanocrystals which comprise a plurality of polynucleotide strands ofknown sequence. In a * more preferred embodiment, the plurality ofpolynucleotide strands are operably linked to the functionalizednanocrystals such that all, or a substantial amount, of one terminalportion of each polynucleotide strand is bound to the functionalizednanocrystal, and wherein the portion of each polynucleotide strand whichis not bound to the functionalized nanocrystal extends outwardly fromthe functionalized nanocrystal, as will be more apparent from thefigures and following description.

A basic principle of the method and system for signal amplificationusing the compositions according to the present invention is that amolecular probe is used to detect a target molecule, if present, by thebinding specificity of the molecular probe for the target molecule or aportion thereof; and the molecular probe is, or becomes, operably linkedto a primary dot, and successive additions of secondary dots and primarydots result in the formation of a dendrimer, wherein the fluorescence ofthe primary dots and secondary dots of the dendrimer provide the signaland signal amplification in detecting the binding of the molecular probeto the target molecule. In one embodiment of the present invention, aneffective amount of molecular probe is first contacted with a sampleunder suitable conditions of a reaction for the molecular probe tocontact and bind its target molecule, if present, in the sample beinganalyzed for the presence or absence of the target molecule.Subsequently, an effective amount of primary dots, which have bindingspecificity for the molecular probe, is added to the reaction. Inanother embodiment of the present invention, the molecular probe isfirst operably linked to primary dots, and then an effective amount ofthe molecular probe-primary dot complex is contacted with the sampleunder conditions suitable for the molecular probe to contact and bindits target molecule, if present, in the sample being analyzed for thepresence or absence of the target molecule. In a further embodiment, themolecular probe is synthesized as a part of the primary dots, and hence,an effective amount of primary dots is contacted with a sample underconditions suitable for the portion comprising the molecular probe tocontact and bind its target molecule, if present, in the sample beinganalyzed for the presence or absence of the target molecule. In thislatter embodiment, the molecular probe may be incorporated as a coatwhich is made part of the nanocrystal portion of the primary dots, orsynthesized as part of the polynucleotide stands of the primary dots.Applicable to any of these embodiments, (a) the primary dots are addedin aneffective amount, and (b) to the primary dots are added, undersuitable conditions for contact and hybridization, an effective amountof a species of functionalized nanocrystals (“secondary dots”) havingpolynucleotide strands comprised of sequence complementary to thesequence of the polynucleotide strands of the primary dots (note, thatthe terms “primary dots” and “secondary dots” are used only for purposesof brevity of description and illustration). Optionally, a wash step maybe performed prior to the addition of the secondary dots to the primarydots so as to remove any primary dots in the system that have becomenon-specifically associated with a target molecule, if present. Undersuitable conditions, an effective amount of secondary dots will contactand hybridize (via complementary strands) to the primary dots in forminga dendrimer. In multiple steps in which subsequent additions of aneffective amount of functionalized nanocrystals alternate betweenprimary dots and secondary dots, a dendrimer of multiple layers offunctionalized nanocrystals is formed, thereby resulting in detectablesignal and an exponential increase in the amount of detectable signalfor detecting a molecular probe bound to a target molecule.

The compositions according to the present invention comprisefunctionalized nanocrystals which are (a) water-soluble fluorescentnanocrystals, and (b) comprise a plurality of polynucleotide strands ofknown (predetermined) sequence. Desirable features of the basicfluorescent nanocrystals themselves include that they can be excitedwith a single excitation light source resulting in a detectablefluorescence emission of high quantum yield (e.g., a single nanocrystalhaving at a fluorescence intensity that may be a log or more greaterthan that a molecule of a conventional fluorescent dye) and with adiscrete fluorescence peak. The nanocrystals typically should have, asubstantially uniform size of less than 200 Angstroms, and preferablyhave a substantially uniform size in the range of sizes of from about 1nm to about 5 nm, or less than 1 nm.

In that regard, illustrative fluorescent nanocrystals are preferablycomprised of a core of Cdx wherein X is Se or Te or S. Such quantum dotsare well known in the art. CdX quantum dots can be passivated with anoverlayering (“shell”) uniformly deposited thereon. A preferredpassivating shell is comprised of YZ wherein Y is Cd or Zn, and Z is S,or Se. Quantum dots having a Cdx core and a YZ shell have also beengenerally described in the art. However, a feature of the quantum dotsused to operably link to a plurality of polynucleotide strands is thatthe quantum dots have been functionalized to be water-solublenanocrystals, and have reactive functionalities to operably link thenanocrystals to a plurality of polynucleotide strands. “Water-soluble”is used herein to mean that the nanocrystals are sufficiently soluble orsuspendable in a aqueous-based solution including, but not limited to,water, water-based solutions, buffer solutions, that are used indetection processes, as known by those skilled in the diagnostic art.

A mercaptocarboxylic acid coating imparts some water solubility to thequantum dots (Chen and Nie, 1998, Science 281:2016-2018), whereasquantum dots capped with trialkylphosphine oxide are soluble only inorganic, non-polar (or weakly polar) solvents. Another method to makethe CdX core/YZ shell quantum dots water-soluble is by the formation ofa layer of silica around the dots (Bruchez et al., 1998, Science 281:2013-2015). However, depending on the nature of the coating group,quantum dots which have been reported as water-soluble may have limitedstability in an aqueous solution, particularly when exposed to air(oxygen) and/or light. More particularly, oxygen and light can cause themolecules comprising the coating to become oxidized, thereby formingdisulfides which destabilize the attachment of the coating molecules tothe shell. Thus, oxidation may cause the coating molecules to migrateaway from the surface of the quantum dots, thereby exposing the surfaceof the quantum dots in resulting in “destabilized quantum dots”.Destabilized quantum dots form aggregates when they interact together,and the formation of such aggregates eventually leads to irreversibleflocculation of the quantum dots. Additionally, carboxylate groups cancause non-specific binding, particularly to one or more molecules in asample other than the target molecule, which is not desirable in adetection assay. Described herein are functionalized nanocrystalscomprising a plurality of polynucleotide strands to provide signal andsignal amplification.

Example 1

In this embodiment is illustrated the production of exemplary, basicfluorescent nanocrystals appropriate for functionalization according tothe present invention. The following examples are illustrative of themethods and functionalized nanocrystals of the present invention. Asdisclosed in detail in U.S. Pat. No. 6,114,038 (the disclosure of whichis herein incorporated by reference), fluorescent nanocrystals comprisenanocrystals which have been functionalized by the addition of aplurality of molecules; and preferably, the molecules are selected froman amino acid, a carboxylic acid, and a combination thereof. Forexample, the nanocrystals may comprise semiconductor nanocrystals thathave a core selected from the group consisting of CdSe, CdS, and CdTe(collectively referred to as “Cdx”), and may further comprise apassivating shell comprised of YZ wherein Y is C4 or Zn, and Z is S, orSe. In one preferred embodiment, the CdX core/YZ shell nanocrystals aretreated with a large excess of mercaptocarboxylic acid in exchanging thetrialkylphosphine oxide coat with a coat comprising a plurality ofcarboxylic acid molecules. For example, (CdSe)ZnS nanocrystals wereprepared in a pyridine solution. The pyridine overcoating of the (Cdx)core/YZ shell nanocrystals were exchanged with a carboxylic acidcomprising mercaptocarboxylic acid. Exchange of the coating group isaccomplished by treating the water-insoluble, pyridine-cappednanocrystals with a large excess of neat mercaptocarboxylic acid. Toaccomplish this, the pyridine-capped (CdSe)ZnS nanocrystals wereprecipitated with hexanes, and then isolated by centrifugation. * Theresidue was dissolved in neat mercaptoacetic acid, with a few drops ofpyridine added, if necessary, to form a transparent solution. Chloroformwas added to precipitate the nanocrystals and wash away excess thiol.The nanocrystals were isolated by centrifugation, washed once more withchloroform, and then washed with hexanes. The residue was briefly driedwith a stream of argon. The resultant nanocrystals, coated withmolecules of carboxylic acid, were then soluble in water or otheraqueous solutions. The nanocrystals, in an aqueous solution, werecentrifuged once more, filtered through a 0.2 μm filter, degassed withargon, and stored in an amber vial. The nanocrystals may then be furtherfunctionalized by an amino acid. For example, diaminocarboxylic acidmolecules were operably bound to the carboxylic acid molecules of thenanocrystals by using commercially available crosslinking agents andmethods known to those skilled in the art. As illustrated in FIG. 1, thecarboxylic acid-coated nanocrystals were dissolved in an aqueous buffersystem (pH of about 7). To the nanocrystals was added EDC(1-ethyl-3-(3-dimethylaminopropyl carbodimide) and sulfo NHS(sulfo-N-hydroxysuccinimide) in 500-1000 times excess. The resultingsolution was stirred at room temperature for 30 minutes. Mercaptoethanolwas added to neutralize unreacted EDC at 20 mM concentration and stirredfor 15 minutes. The entire solution was then added drop-wise, withstirring, to a solution of a diaminocarboxylic acid comprising lysine(large excess) in the same buffer; and the mixture was stirred for 2hours at room temperature. Ethanolamine (30 mM) was added to quench thereaction; and the mixture was stirred for 30 minutes at room temperatureor left overnight at 4° C. The solution was centrifuged to remove anyprecipitated solids, and then ultrafiltered through a 3 OkD MWcentrifugal filter. The resultant concentrated, fluorescent nanocrystalscan be solubilized in an aqueous solution of choice. Once solubilized,the resulting solution can be stored in an amber vial under an inert gasto prevent flocculation. The fluorescent nanocrystals may be operablybound to a successive layer of amino acid molecules by, for example,repeating the procedure and reaction using EDC and sulfoNHS with theamino acid molecules comprising the successive layer.

Similarly, a nanocrystal comprising a doped metal oxide nanocrystal maybe operably bound to a plurality of molecules (e.g., a carboxylic acid,and amino acid, or a combination thereof) using methods known in theart. For example, the plurality of molecules having reactivefunctionalities comprising free carboxyl groups can be chemi-sorbed,adsorbed or otherwise permanently added to the metal oxide portion ofthe nanocrystal. For example, the metal oxide nanocrystals are suspendedin an aqueous solution of an amino acid comprising homocysteine having apH of about 3.5 for about an hour. The reaction is then stopped byadjusting the pH to neutral, and dialyzing out the aqueous solution.

As an alternative, fluorescent nanocrystals may be capped andfunctionalized with a plurality of amino acid molecules. For example,nanocrystals (e.g., (CdSe)ZnS) coated with an organic layer (e.g.,mercaptoacetic acid) were treated with a molar excess of homocysteine inreplacing the organic layer with a coating comprising a plurality ofamino acid molecules. The approximate number of surface Zn sites on thespecific size of nanocrystals utilized was calculated. At least a 5times molar excess of homocysteine (as compared to the number of surfaceZn sites) was added to the nanocrystals, as per the following formula.*Grams homocysteine=5(number of Zn surface sites) (volume of solutioncontaining the nanocrystals) (concentration of nanocrystals in solution)(135.2).

The mixture was stirred to dissolve the homocysteine, and then stored at4° C. for 24 hours. The resultant solution was then centrifuged toremove any precipitate, and the supernatant was transferred to acentrifugal filter for the appropriate volume of supernatant(preferably, with a molecular weight cutoff of about 10 kD or below toretain the fluorescent nanocrystals coated with homocysteine). Aftercentrifugation, and when the desired minimum volume is reached, thefluorescent nanocrystals were then rediluted in the appropriate aqueoussolution (e.g., HEPES buffer) to a volume in which the original mass ofhomocysteine had been dissolved. The steps of filtering and redilutionof the fluorescent nanocrystals in solution may be repeated to improvepurity. The resultant fluorescent nanocrystals comprisinghomocysteine-coated nanocrystals may then be degassed by bubbling withan inert gas, and then stored at 4° C. in an amber bottle.

Example 2

Illustrated in this Example is a preferred embodiment of exemplaryfunctionalized nanocrystals. The functionalized nanocrystals wereproduced using the methods illustrated in Example 1. In a preferredembodiment, the fluorescent nanocrystals were functionalized by operablylinking diaminocarboxylic acid to the fluorescent nanocrytsals (e.g., tothe capping compound) in forming functionalized nanocrystal. In apreferred embodiment, the diaminocarboxylic acid (a) contributes to thewater-solubility of the functionalized nanocrystal because it has polarfunctional groups which can hydrogen-bond with water; (b) has at leasttwo free functional groups which are carboxyl-reactive, thereby enablingthe diaminocarboxylic acid molecule to operably link to, and maycrosslink, carboxyl groups extending from the capping compound on thecapped fluorescent nanocrystals; (c) once operably linked to the cappingcompound, has one or more free functional groups which can be used foroperably linking to one or more polynucleotide strands (or to a linker);and (d) provides other beneficial properties to the resultantfunctionalized nanocrystals. A preferred diaminocarboxylic acidcomprises lysine. The resultant concentrated, functionalizednanocrystals can be solubilized in an aqueous solution of choice.

Regarding stability, as shown in FIG. 2, the functionalized nanocrystalscontaining a coat of diaminocarboxylic acid (“FN”) unexpectedly show asignificant increase in stability in an aqueous environment compared toquantum dots having an outer layer of just the capping compound (“W-SN),when exposed over time to identical conditions of an oxidizingenvironment (e.g., light and air). Additionally, as shown in FIG. 3,functionalized nanocrystals containing a coat of diaminocarboxylic acid(“FN”) unexpectedly result in a significant decrease in non-specificbinding compared to quantum dots having an outer layer of just thecapping compound (“WSN), when each were contacted with a surface that isboth hydrophilic and hydrophobic (e.g., as may be encountered in adetection system), followed by washing of the surface, followed bydetection of residual nanocrystals (as measured by number of events offluorescence versus the intensity of fluorescence; using a fluorescencemicroscope with a video camera attachment, time of exposure- 1/30th of asecond).

In another embodiment, as also illustrated in FIG. 1, the functionalizednanocrystals are further functionalized by operably linking the coatingcomprising diaminocarboxylic acid to a plurality of polynucleotidestrands; or to a combination comprising a linker which is then operablylinked to the molecular probe, and a plurality of polynucleotidestrands. For example, depending on factors such as the species and/oramount of amino acid (e.g., diaminocarboxylic acid) used to operablylink to the capping compound, each operably linked amino acid may haveone or more reactive functionalities (e.g., free amino group, freecarboxyl group, and a combination there-of) that can be used to operablylink to a reactive functionality of a polynucleotide strand (or to alinker). As an illustrative example, polynucleotide strands having freecarboxyl-reactive groups (e.g., amine groups) can be operably linked tofree carboxyl groups of the molecules of diaminocarboxylic acidcomprising a coating of the functionalized described herein). In analternative, polynucleotide strands having free amino-reactive groups(e.g., carboxyl groups) can be operably linked to free amino groups ofthe molecules of diaminocarboxylic acid comprising a coating of thefunctionalized nanocrystals using methods known in the art. Ifdesirable, essentially the same procedure can be used to operably linkan additional amino acid layer onto the diaminocarboxylic acid layer,and then to operably link the resultant functionalized nanocrystal to aplurality of polynucleotide strands (or linker).

To illustrate this embodiment, molecules representative ofpolynucleotide strands having free carboxyl-reactive groups (aminegroups) were operably linked to the functionalized nanocrystals usingthe methods summarized herein. Briefly, functionalized nanocrystals (1ml, 8.1×10⁻⁹ mol) were esterified by treatment with EDC (8.1×10⁻⁶ mol),followed by treatment with sulfo-NHS (8.9×10⁻⁶ mol) at ambienttemperature in buffered aqueous solution (at about pH 7.4) for 30minutes. 2-mercapto-ethanol was added to the solution at a concentrationof 20 mM, and the mixture was stirred for 15 minutes to quench anyunreacted EDC. The nanocrystals were then contacted with a molconcentration of molecules (depending on the size, and desired number)for operably linking a plurality of the molecules to the functionalizednanocrystals, and the reaction mixture was stirred for 2 hours (e.g., orreacted in other suitable conditions for forming an amide bond betweenthe EDC activated carboxylates of the diaminocarboxylic acid layer andthe amine groups on the polynucleotide strands). Ethanolamine was addedat a concentration of 30 mM to quench the coupling reaction, and thereaction mixture was stirred for 30 minutes. The resulting solution wasthen filtered to remove excess reagents. The concentrated material wasthen diluted to 1 ml in buffer (e.g., PBS) or other suitable aqueoussolution.

Example 3

This Example illustrates additional embodiments comprisingfunctionalized nanocrystals comprising a plurality of polynucleotidestrands. The polynucleotide strand is a nucleic acid molecule which istotally or substantially single-stranded and has no particular size,length or content limitations, so long as the polynucleotide strand canfulfill its purpose in dendrimer formation. The nucleic acid compositionof the polynucleotides may be selected from molecules which include, butare not limited to, ribonucleotides (RNA), deoxyribonucleotides (DNA),RNA/DNA hybrids, naturally occurring nucleotides, and synthetic ormodified nucleotides (e.g., oligonucleotide analogs, backbone modifiedoligonucleotide analogs, and morpholino-based polymers). A preferredlength of the polynucleotide strand is determined from such factors asthe nucleic acid composition (nucleobase type; e.g., analog or naturallyoccurring), the desired specificity of annealing to its complementarystrand, the sequence of nucleobases, and desired annealing temperatures.

In a preferred embodiment, the length of the polynucleotide strand is anumber of nucleobases wherein the number is between about 6 and about50. In a more preferred embodiment, the length of the polynucleotidestrand is a number of nucleobases wherein the number is between about 10and about 20. As shown in SEQ LID Nos: 1-4, a preferred length is about18 nucleobases.

The preferred content requirement for a polynucleotide strand is that itbe comprised of a known (predetermined) nucleotide sequence. Thus,functionalized nanocrystals have extending therefrom a plurality ofpolynucleotide strands of known sequence. It is apparent to thoseskilled in the art that the polynucleotide strand may be comprised of aplurality of a single, repeated nucleobase; or a plurality a combinationof nucleobases. For example, such a sequence can be made up of acombination of nucleobases (e.g., of more than one type of nucleobase,wherein the nucleobase types comprise A, T, C, G, and U). However, in apreferred embodiment, the known sequence substantially comprises (about60% to about 100%) of a single type of nucleobase. In illustrating thispreferred embodiment, SEQ ID NO: 1 is a polynucleotide strand sequencesubstantially comprised of A; SEQ ID NO: 2 is a polynucleotide strandsequence substantially comprised of T; SEQ ID NO: 3 is a polynucleotidestrand sequence substantially comprised of G; and SEQ ID NO: 4 is apolynucleotide strand sequence substantially comprised of C. Formationof dendrimers requires that a species of functionalized nanocrystalscomprising a plurality of polynucleotide strands (“primary dots”) havepolynucleotide strands comprised of known sequence that arecomplementary in sequence to the polynucleotide strands of anotherspecies of functionalized nanocrystals (“secondary dots”). For purposesof illustration, but not limitation, a primary dot comprises a pluralityof polynucleotide strands, each strand substantially comprising polyT(e.g., SEQ ID NO: 31); and a secondary dot comprises a plurality ofpolynucleotide strands, each strand substantially comprising polyT(e.g., SEQ ID NO:2). In another non-limiting illustration, a primary dotcomprises a plurality of polynucleotide strands, each strandsubstantially comprising AG repeats (e.g., SEQ ID NO:5); and a secondarydot comprises a plurality of polynucleotide strands, each strandsubstantially comprising TC repeats (e.g., SEQ ID NO:6).

In addition to the preferred embodiment illustrated in Example 2 herein,using techniques known in the art, a plurality of polynucleotide strandsmay be operably linked to functionalized nanocrystals in formingfunctionalized nanocrystals comprising a plurality of polyunucleotidestrands according to the present invention. Preferably, eachpolynucleotide strand is operably linked to the functionalizednanocrystal such that contact is substantially between a single terminusof the polynucleotide strand and a coating of the functionalizednanocrystal. Such arrangement provides for the polynucleotide extendingoutwardly from the resultant functionalized quantum dot, optimallyallows for a plurality of polynucleotide strands to be attached to thefunctionalized nanocrystal, and may provide a minimum of quenching oflight emission when such fluorescence emission is emitted by an excitedfunctionalized nanocrystal. To operably link each polynucleotide strandto a functionalized nanocrystal, utilized is one or more reactivefunctionalities.

In another preferred embodiment, carboxylate-terminated capped quantumdots are produced, such as by the method illustrated in Example 1herein, or a similar method. The carboxylate groups of the cappingcompound and a carboxyl-reactive group of a linker are reacted by ameans which catalyzes the formation of a chemical association or bondformation between the carboxylate groups and the carboxyl-reactivegroups. As an example, using methods similar to that illustrated inExample 2 herein, EDC was used to operably link a linker comprising amolecule having binding specificity for biotin to capped fluorescentnanocrystals in forming functionalized nanocrystals. Molecules havingbinding specificity for biotin are known in the art to include, but arenot limited to, avidin, streptavidin, and derivatives or modifiedversions thereof; e.g., neutravidin, nitro-avidin, nitro-streptavidin,aceylated avidin, and the like. For purposes of brevity of description,but not limitation, a linker having biotin-binding specificity will bereferred to hereinafter as “avidin”. In an illustrative embodiment,fluorescent nanocrystals were capped by a coating with11-mercaptoundecanoic acid, and deprotonated with potassium-t-butoxide.The carboxylated fluorescent nanocrystals were then esterified bytreatment with EDC followed by sulfo-N-hydroxy-succinimide (sNHS). Thesefluorescent nanocrystals were then contacted with avidin undersufficient conditions to form an amide bond between the EDC-activatedcarboxylate of the capped fluorescent nanocrystals and the amine groupson avidin; thereby forming functionalized nanocrystals that wereavidinylated. The avidinylated, functionalized nanocrystals may then becontacted with, and operably linked to, a plurality of polynucleotidestrands, each of which contains one or more biotin molecules (comprisingnative biotin, or a biotin derivative having avidin-binding activity;e.g., biotin dimers, biotin multimers, carbo-biotin, and the like).Preferably, each of the plurality of polynucleotide strands arebiotinylated at a single terminus of the polynucleotide strand. Usingmethods known to those skilled in the art, biotin molecules can be addedto or incorporated in a polynucleotide strand, and even localized to oneterminus, such as by directing synthesis of the polynucleotide strandswith nucleotides and biotinnucleotides, or by biotinylating the 5′aminogroup of the polynucleotide with sulfo-NHS-biotin. Thus, bycontacting avidinylated, water-soluble fluorescent nanocrystals withbiotinylated polynucleotide strands, formed is a functionalizednanocrystal comprising a plurality of polynucleotide strands extendingtherefrom.

In another preferred embodiment, the functionalized nanocrystals of thepresent invention are produced using reactive functionalities comprisingthiol group and thiol reactive groups. One illustration of thisembodiment involves use of maleimide derivatives. For example, CdXcore/YZ shell quantum dots are capped by a coating withmercapto-functionalized amines or amino acid (e.g.,aminoethanethiol-HCl, homocysteine, or1-amino-2-methyl-2-propanethiol-HCl). Thus, the cap comprises reactivefunctionalities comprising amine groups. To these capped fluorescentnanocrystals are added (either in the presence or absence of EDC) amaleimide derivative that reacts with the free amine groups. Such amaleimide derivative may include, but is not limited to3-maleimidopropionic acid N-hydroxy-succinimide ester,3-maleimidopropionic acid, 3-maleimidobenzoic acid N-hydroxy-succinimideester, 4-(maleimido-methyl)-1-cyclohexanecarboxylic acidN-hydroxy-succinimide ester. The resultant functionalized nanocrystals,having thiol-reactive groups, can interact with and bind to a pluralityof polynucleotide strands, wherein each polynucleotide strand has beenpreviously derivatized with one or more thiol groups, using methodsknown in the art for operably linking a thiol group to a thiol-reactivegroup. Generally, it is preferred that the polynucleotide strand bederivatized substantially at either its 3′ or 5′ terminus (“capped”)with the thiol group. Exemplary means for capping one or more thiolgroups to nucleotides are known in the art (see, e.g., U.S. Pat. Nos.5,811,534, 5,663,242, 5,441,867, and 5,412,087, the disclosures of whichare herein incorporated by reference). Thus, formed are functionalizednanocrystals comprising a plurality of polynucleotide strands extendingtherefrom.

In another preferred embodiment, the functionalized nanocrystals of thepresent invention are produced utilizing reactive functionalitiescomprising amine groups and amine reactive groups. One illustration ofthis embodiment involves capping CdX core/YZ shell quantum dots with acoating comprising mercapto-functionalized amines (e.g.,amino-ethanethiol-HCl, homocysteine, or1-amino-2-methyl-2-propanethiol-HCl). Thus, the cap comprises reactivefunctionalities with amine groups. These capped fluorescent nanocrystalsare contacted, and operably linked, to a plurality of polynucleotidestrands, wherein each polynucleotide strand has been previouslyderivatized with one or more amino reactive groups. Amino reactivegroups are known to those skilled in the art to include, but are notlimited to, active ester groups, haloacetyl groups, azide groups,isocyanate groups, isothiocyanate groups, and acid anhydride groups(see, e.g., U.S. Pat. No. 5,580,923, the disclosure of which is hereinincorporated by reference). Exemplary means for capping one or moreamino reactive groups to nucleotides are known in the art previously(e.g., attachment of an ester to the 3′ or 5′ terminus of anoligonucleotide, U.S. Pat. No. 5,639,604; attachment of an aryl azide,U.S. Pat. No. 5,700,921; the disclosures of which are hereinincorporated by reference).

Example 4

This example illustrates various embodiments of using functionalizednanocrystals comprising a plurality of polynucleotide strands extendingtherefrom in a method of detection of a target molecule using the signalgeneration and signal amplification afforded by the dendrimer formationaccording to the present invention. In a preferred embodiment,functionalized nanocrystals having extended therefrom a plurality ofpolynucleotide strands of known sequence comprise primary dots, and toeach primary dot is operably linked to a molecular probe. As apparent toone skilled in the art from the descriptions herein, the operablylinking of the primary dot to the molecular probe can be done in amanner selected from the group consisting of prior to the addition ofthe molecular probe to a sample being analyzed for a target molecule,after the molecular probe has been added to the sample being analyzedfor a target molecule, and a combination thereof. As previouslydescribed in more detail herein, the molecular probe may be any probeused in a detection system to detect the presence or absence of adesired target molecule for which the molecular probe has bindingspecificity and avidity.

In one illustrative embodiment, and as shown in FIG. 3, the molecularprobe may be a mAb. In one variation of this embodiment, the primarydots are first reacted with, and operably linked to, molecular probeprior to the addition of an effective amount of the primarydot-molecular probe complex to a sample being analyzed for the presenceor absence of a target molecule. In another illustrative embodiment, andin a detection system, molecular probe is first added in an effectiveamount to a sample being analyzed for the presence or absence of atarget molecule in suitable conditions for the molecular probe tocontact and bind to target molecule if present in the sample.Optionally, a wash step may be performed to remove from the detectionsystem any unbound or nonspecifically bound molecular probe. Primarydots are added in an effective amount to contact and operably link withmolecular probe that may be present in the detection system. In eitherof these illustrative embodiments, the terminal portions of thepolynucleotide strands that are not bound to the primary dots, and thatextend outwardly from the functionalized nanocrystal (“free ends”)comprise one or more reactive functionalities (e.g., a linker) that maybe used to contact and operably link to one or more reactivefunctionalities associated with the mAb. For example, as previouslydescribed herein and using methods known to those skilled in the art,biotin molecules can be added to or incorporated in a polynucleotidestrand so as to be localized to one terminus (e.g., by incorporatingbiotin-deoxynucleotides during synthesis, or by biotinylating the 5′aminogroup of the polynucleotide with sulfo-NHS-biotin). A mAb (or othermolecular probe comprising a protein such as antibody molecule, lectin,and the like) may be attached (conjugated, coupled, etc.) to avidinusing methods known to those skilled in the art. Such attachment shouldtake place in a portion of the molecular probe which does notsubstantially affect the binding of the molecular probe to its ligand.For example, mAb and avidin may be conjugated with a thio-ether linkageusing methods known in the art. Thus, the free ends of thepolynucleotide strands comprising biotin are contacted with and operablylinked to the avidinylated mAb in operably linking the functionalizednanocrystals to the molecular probes. This biotin-avidin system may beused to either operably link the primary dots with molecular probe priorto the addition of the molecular probe to a sample being analyzed forthe presence or absence of a target molecule; or to operably link theprimary dots to the molecular probe after the molecular probe hasalready been added to the sample being analyzed; or a combinationthereof. As shown in FIG. 3, after the molecular probe and primary dot(1′) have been added, successive additions of secondary dots (2′)(having polynucleotide strands complementary in sequence to thepolynucleotide strands of the primary dots) and primary dots may beadded to form dendrimers.

In another illustrative embodiment, and as illustrated in FIGS. 4A and4B, the functionalized nanocrystals comprising a plurality ofpolynucleotide strands are operably linked to the molecular probe viaone or more reactive functionalities, wherein the operably linking takesplace between a coating of the functionalized nanocrystals (other thanthe polynucleotide strands) and the molecular probe. For example, aspreviously described herein in more detail, functionalized nanocrystalsmay be avidinylated, and then reacted in a controlled manner so as tooperably link a finite number of a plurality of biotinylatedpolynucleotide strands (e.g., by limiting the ratio of polynucleotidestrands: functionalized nanocrystals in the reaction for producingfunctionalized nanocrystals comprising a plurality of polynucleotidestrands). Generally, each avidin molecule can bind up to 4 biotinmolecules. Thus, by limiting the number of biotinylated polynucleotidestrands operably linked thereto, a functionalized nanocrystal may stillhave avidin capable of binding one or more biotin molecules, such as maybe present on a molecular probe. Therefore, using methods known to thoseskilled in the art, the molecular probe may be biotinylated.Biotinylation of oligonucleotides has been previously described herein.Biotinylated molecular probes comprising proteins (e.g., lectins, mAbs,etc.) are commercially available, or can be produced by one of severalmethods known in the art such as derivatization of the protein vialysine e-amino groups, or via thiol groups generated by reduction ofcysteines. Thus, as illustrated in FIG. 4A (illustrating a molecularprobe comprising a biotinylated (“B”) nucleic acid molecule; e.g., anoligo probe) and in FIG. 4B (illustrating a molecular probe comprising abiotinylated (“B”) antibody), an avidin (“A”) of the primary dots (1′)is contacted with and operably linked to the biotinylated (“B”)molecular probe. This biotin-avidin system may be used to eitheroperably link the primary dot with molecular probe prior to the additionof the molecular probe to a sample being analyzed for the presence orabsence of a target molecule, or to operably link the primary dot to themolecular probe after the molecular probe has already been added to thesample being analyzed. As shown in FIGS. 4A and 4B, after the molecularprobe and primary dot (1′) have been added, successive additions ofsecondary dots (2′) (having polynucleotide strands complementary insequence to the polynucleotide strands of the primary dots) and primarydots may be added to form a dendrimer.

In another illustrative embodiment, and using the methods illustratedabove, free ends of polynucleotide strands may comprise one or morereactive functionalities used to operably link molecular probe. In thisembodiment, preferably the primary dots are operably linked to themolecular probe prior to the addition of the molecular probe to thesample being analyzed for the presence or absence of target molecule.For example, and as previously described in more detail herein, aterminal portion of a polynucleotide strand may be capped with one ormore thiol groups. A molecular probe may be derivatized to contain oneor thiol-reactive groups using methods known to those skilled in the art(e.g., using a hetero-bifunctional crosslinking reagent such asSMCC-succinimidyl 4-(N-maleimidomethypcyclohexane-1-carboxylate; orSPDP-succinimidyl 3-(2-pyridyldithio) propionate). Under suitableconditions, contacting the thiol groups contained on the free ends ofthe polynucleotide strands with the thiolreactive groups may result inoperably linking the primary dots to the molecular probes. The resultantprimary dot-molecular probe complex may then be added to the sample.Successive additions of secondary dots and primary dots may then beadded in forming dendrimers if target molecule is present. Alternately,and similarly as described above, the quantum dots are capped withmercapto-functionalized amines; the capped quantum dots arefunctionalized by the addition of a maleimide derivative that reactswith amino groups of the capping compound; the functionalizednanocrystals are operably linked to thiolderivatized polynucleotidestrands in a controlled manner so as to bind a finite number of aplurality of thiolated polynucleotide strands; and the remaining freethiol-reactive groups operably link to thiolated molecular probe informing thioether bonds between the primary dots and molecular probe. Amolecular probe, such as one comprised of protein (e.g., antibody,lectins, peptides, and the like) may be derivatized to contain thiolgroups, using methods known to those skilled in the art. For example, athiol group may be generated by a partial reduction of the protein.Thus, the primary dot comprises a plurality of polynucleotide strands,and further comprises one or more molecules of molecular probe. Theresultant primary dots may then be added to a sample being analyzed forthe presence or absence of a target molecule, followed by successiveadditions of secondary dots and primary dots. The primary dots added inthe successive additions may have or lack molecular probe operablylinked thereto.

In another illustrative embodiment, and as shown in FIG. 5, a linker maybe used to contact and operably link a polynucleotide strand of theprimary dot to a molecular probe. For example, as previously describedherein and using methods known to those skilled in the art, biotinmolecules can be added to or incorporated in a polynucleotide strand soas to be localized to the free end of the polynucleotide strand.Likewise, a molecular probe can be biotinylated (e.g., a biotinylatedoligonucleotide probe, a biotinylated mAb, etc.). As illustrated in FIG.5, avidin (“A”) is used as a linker to operably link the biotinylated(“B”) polynucleotide strands of primary dots to biotinylated (“B”)molecular probe, as generally, avidin can bind multiple molecules ofbiotin. It will be apparent to those skilled in the art that there areseveral ways in which avidin may be used as a linker. For example, theavidin may be first added to the molecular probe (either in the sample,or before added to the sample), and then the primary dots are added; orthe avidin may be first added to the primary dots before adding theprimary dots to the molecular probe; or the avidin may be addedsimultaneous with the mixture of the primary dots with the molecularprobe. In embodiments in which the avidin is first added to either ofthe primary dots or the molecular probe, a wash step may be performed toremove any unbound avidin before the next component is added. As shownin FIG. 5, after addition of the biotinylated (“B”) primary dots (1′),avidin (“A”), and the biotinylated (“B”) molecular probe, successiveadditions may be made of secondary dots (2′) and primary dots (1′).

In another illustrative embodiment, and as shown in FIGS. 6A, 6B, and 7,a linker may be used to contact and operably link a functionalizednanocrystal to a molecular probe. For example, where both entities (thepolynucleotide strand of the primary dots, and molecular probe) arethiolated, a homobifunctional linker may be used such asbismaleimidohexane. Where one entity has a reactive functionalitycomprising one or more thiol groups, and the other entity has a reactivefunctionality comprising one or more amino groups, heterobifunctionallinkers may include, but are not limited to, sulfo-GMBS, sulfo-MBS,sulfo-SMCC, and sulfo-SMPB. In another variation of this embodiment, themolecular probe comprises an oligonucleotide probe which is firstoperably linked to the primary dots, and then added to the sample beinganalyzed for the presence or absence of the target molecule underconditions which promote contact and binding of the molecular probe tothe target molecule, if present. Alternatively, as shown in FIG. 7, thelinker may be a nucleic acid molecule which has, at one end comprising aterminal portion, a sequence complementary (for hybridizing) to thesequence of a terminal portion of the free end of a polynucleotidestrand of the primary dot; and an opposite end or terminal portion whichhas a sequence which is sufficiently complementary (for hybridizing) toa portion of the molecular probe comprising an oligonucleotide. In thislatter variation, conditions suitable for contact and hybridization maybe used to either operably link the primary dot with molecular probeprior to the addition of the molecular probe to a sample being analyzedfor the presence or absence of a target molecule, or to operably linkthe primary dot to the molecular probe after the molecular probe hasalready been added to the sample being analyzed under suitableconditions for the molecular probe to contact and bind to the targetmolecule, if present in the sample.

In another embodiment, as illustrated in FIG. 8, the molecular probecomprising an oligonucleotide is synthesized as part of the free end, atthe terminal portion, of one or more of the plurality of polynucleotidestrands of the primary dots, using methods known to those skilled in theart of nucleic acid molecule synthesis. This embodiment requires knowingthe sequence of the oligonucleotide probe so as to be able to synthesizeit as part of the polynucleotide strand; and thus, is unlike most of theother embodiments described herein for operably linking the molecularprobe to the primary dots which rely on reactive functionalities otherthan nucleic acid complementarity. An effective amount of the primarydot of this embodiment is contacted with a sample under suitableconditions for the portion comprising the molecular probe to contact andbind (e.g., hybridize) to its target molecule being analyzed for thepresence or absence of the target molecule.

In summary, primary dots may be operably linked to molecular probe usingmeans which include, but are not limited to, (a) by a reactivefunctionality on one or more of the plurality of polynucleotide strandsand a reactive functionality associated with the molecular probe (see,e.g., FIG. 3); (b) by a reactive functionality on the coating of theprimary dot and a reactive functionality associated with the molecularprobe (see, e.g. FIGS. 4A and 4B); (c) by a linker which has one portionthat binds to a reactive functionality on one or more polynucleotidestrands, and another portion which binds to a reactive functionalityassociated with the molecular probe (see, e.g., FIG. 5); (d) by a linkerwhich has one portion that hybridizes to one or more polynucleotidestrands, and another portion which hybridizes to the molecular probecomprising a nucleic acid molecule (e.g., oligonucleotide) (see, e.g.,FIG. 7); and (e) by synthesizing the molecular probe as part of one ormore polynucleotide strands (see, e.g., FIG. 8).

Example 5

In this example, illustrated are embodiments of the method of usingfunctionalized nanocrystals comprising a plurality of polynucleotidestrands according to the present invention for signal generation andsignal amplification for detecting the presence or absence of a targetmolecule. The method uses successive additions of primary dots andsecondary dots to a sample, wherein molecular probe bound to targetmolecule anchors primary dots and secondary dots in forming dendrimersthat provide signal generation and amplification. In a preferredembodiment, “successive additions of primary dots and secondary dots”comprises at least one addition of each and may range up to, or greaterthan, 50 additions of each. For detecting the presence or absence of atarget molecule in a sample, the method comprises a first step ofoperably linking primary dots to molecular probe in forming primarydot-molecular probe complexes, and then contacting an effective amountof primary dot-molecule probe complexes to the sample under suitableconditions for molecular probe to specifically bind to target molecule,if present in the sample. An alternative is to first contact aneffective amount of molecular probe with the sample under suitableconditions for molecular probe to bind to target molecule, if present;and then contacting the sample with an effective amount of primary dotsunder suitable conditions for primary dots to operably link to molecularprobe, if present in the sample. In either embodiment, the sample isthen contacted with an effective amount of secondary dots under suitableconditions for promoting hybridization of the plurality ofpolynucleotide strands of the second dots to the polynucleotide strandsof the primary dots, if present in the sample. The sample is thencontacted with successive additions of an effective amount of primarydots and an effective amount of secondary dots under suitable conditionsfor promoting hybridization. The primary dots added in the successiveadditions may either lack or possess a reactive functionality (e.g.,primary dots used in the first step may have one or more biotinylatedpolynucleotide strands that operably links to avidinylated molecularprobe; however, the primary dots added after the first step and forforming dendrimers may comprise a plurality of polynucleotide strandsthat lack biotinylation). The sample is then exposed to an excitationlight source that is suitable for exciting the functionalizednanocrystals to emit a fluorescence emission.

Detection by detection means of a signal comprising peak fluorescenceemission, generated by excited functionalized nanocrystals, andamplified in intensity by dendrimer formation, is indicative of thepresence of target molecule in the sample (see, e.g., FIGS. 3-8); andabsence of the signal is indicative of the absence of target molecule inthe sample.

The method may further comprise one or more wash steps after eachaddition of functionalized nanocrystals to the sample to remove from thesample unbound or non-specifically bound functionalized nanocrystals(and unbound or non-specifically bound molecular probe). In a preferredembodiment, the primary dots and secondary dots comprise a uniform sizesuch that after excitation, they each emit a fluorescence emission of anarrow bandwidth, which may be representative of a single color; andhence, the fluorescence intensity is amplified by dendrimer formation.Thus, the method may further comprise quantitating the amount of targetmolecule present in a sample by measuring the fluorescence intensity ofthe signal generated, and relating the amount of fluorescence intensityto an amount of target molecule. As previously described in more detailherein, the primary dots and the secondary dots can be excited with thesame excitation light source. The excitation light source (visible, orUV, or a combination thereof) is preferably in the spectral range offrom about 200 nm to about 500 nm; and in a more preferred embodiment,in a spectral range of from about 300 nm to about 400 nm. In a preferredembodiment, fluorescence peak emission comprises a discrete fluorescencepeak in the spectral range of about 400 nm to about 750 nm. Thefluorescence emission may be detected, or detected and quantitated, byappropriate detection means (e.g., one or more of: photodetector,filter, charge couple device camera (CCD camera) fluorescencemicroscope, fluorescence imaging micro-scope, a scanner or -reader fordetecting fluorescence, a fluorescence cube, a computer for processingdetected fluorescence, and the like).

For example, CdSe/ZnS quantum dots having a substantially uniform sizecomprising a diameter of about 68.4 angstroms (A) may be excited withlight of a spectral range of from about 400 nm to 500 nm, and emit afluorescence peak (orange) at 609 nm. CdSe/ZnS quantum dots having asubstantially uniform size comprising a diameter of about 53.2 A may beexcited with light of a spectral range of from about 400 nm to 500 nm,and emit a fluorescence peak (yellow) at 545 nm. CdSe/ZnS quantum dotshaving a substantially uniform size comprising a diameter of about 46.6A may be excited with light of a spectral range of from about 400 nm to500 nm, and emit a fluorescence peak (green) at 522 nm. In a preferredembodiment, detection may be by detection means comprising a scanner orreader or other analytical instrument which can detect discretefluorescence peaks in the spectral range of about 400 nm to about 750nm; and, optionally (when more than one color is used in the methodaccording to the present invention), distinguish between discretefluorescence peaks within that spectral range. In the class of quantumdots used in the present invention, various sizes of dots can be excitedwith a single wavelength spectrum of light, resulting in many emissionsof discrete fluorescence peaks corresponding to colors that can bedetected simultaneously and distinctly. Thus, for example, it will beapparent to those skilled in the art that method of the presentinvention encompasses detection of more than one target molecule and/oruse of more than one type or specificity of molecular probesimultaneously in the sample, by using a uniform size of functionalizednanocrystals which is different for each type or specificity ofmolecular probe used. For example, a first set of primary dots andsecondary dots comprise a uniform size, wherein the primary dotscomprise a plurality of polynucleotide strands which comprise SEQ IDNO:1, and the secondary dots comprise a plurality of polynucleotidestrands comprising SEQ ID NO:2, and wherein the primary dots areoperably linked to a molecular probe having binding specificity for atarget molecule comprising “gene X”. A second set of primary dots andsecondary dots comprise a uniform size wherein the uniform size isdifferent than the that of the first set (hence, will emit a separate,discrete fluorescence peak which can be distinguished from afluorescence peak emitted from the first set), wherein the primary dotscomprise a plurality of polynucleotide strands which comprise SEQ IDNO:3, and the secondary dots comprise a plurality of polynucleotidestrands comprising SEQ ID NO:4, and wherein the primary dots areoperably linked to a molecular probe having binding specificity for atarget molecule comprising “gene Z”. Hence, a sample may besimultaneously assayed for the presence or absence of gene x and gene Zby using both sets of primary and secondary dots. More particularly, ifboth gene x and gene Z are present, detected simultaneously anddistinctly will be a color representative of the fluorescence emissiongenerated by the functionalized nanocrystals of the first set, and adifferent color representative of the fluorescence emission generated bythe functionalized nanocrystals of the second set, respectively.

Assay kits for the method of the present invention are also provided. Inone preferred embodiment, the assay kit comprises (a) primary dots whichcomprise functionalized nanocrystals comprising a plurality ofpolynucleotide strands which comprise a known sequence, wherein theprimary dots have one or more reactive functionalities; and (b)secondary dots which comprise functionalized nanocrystals comprising aplurality of polynucleotide strands which comprise known sequence whichis complementary to the sequence of the polynucleotide strands of theprimary dots. The reactive functionalities of the primary dots may beused to operably link the primary dots to molecular probe eitherdirectly (i.e., reacts with the reactive functionalities of molecularprobe) or indirectly (reacts with a linker that is used to link theprimary dots to molecular probe). Thus, the kit may further comprise alinker for linking the primary dots to molecular probe. In a preferredembodiment, the primary dots and secondary dots comprise a uniform size.In another embodiment, the primary dots further comprise molecular probewhich is operably linked to the primary dots. The kit may furthercomprise primary dots lacking reactive functionalities. The kit mayfurther comprise a second set of primary dots and secondary dots,wherein the primary dots and secondary dots of the second set comprise auniform size wherein the uniform size is different than the that of thefirst set, wherein the primary dots of the second set comprise aplurality of polynucleotide strands which are complementary in sequenceto the plurality of polynucleotide strands of the secondary dots of thesecond set, and wherein the plurality of polynucleotide strands of theprimary dots of the second set lack sufficient complementarity (thus,will not specifically hybridize) to the plurality of polynucleotidestrands of the primary dots of the first set.

In another preferred embodiment, the assay kit comprises (a) primarydots which comprise functionalized nanocrystals comprising a pluralityof polynucleotide strands which comprise a known sequence, wherein theprimary dots have one or more reactive functionalities; (b) secondarydots which comprise functionalized nanocrystals comprising a pluralityof polynucleotide strands which comprise a known sequence which iscomplementary to the sequence of the polynucleotide strands of theprimary dots; and (c) a molecular probe. In a preferred embodiment, theprimary dots and secondary dots comprise a uniform size. In anotherpreferred embodiment, the molecular probe further comprises one or morereactive functionalities that may be used to operably link the molecularprobe to the primary dots either directly (i.e., reacts with thereactive functionalities of the primary dots) or indirectly (reacts witha linker that is used to link the molecular probe to the primary dots).Thus, the kit may further comprise a linker for linking the primary dotsto molecular probe. The kit may further comprise primary dots lackingreactive functionalities. The kit may further comprise a second set ofprimary dots and secondary dots, wherein the primary dots and secondarydots of the second set comprise a uniform size wherein the uniform sizeis different than the that of the first set, wherein the primary dots ofthe second set comprise a plurality of polynucleotide strands which arecomplementary in sequence to the plurality of polynucleotide strands ofthe secondary dots of the second set, and wherein the plurality ofpolynucleotide strands of the primary dots of the second set lacksufficient complementarity (thus, will not specifically hybridize) tothe plurality of polynucleotide strands of the primary dots of the firstset.

The foregoing description of the specific embodiments of the presentinvention have been described in detail for purposes of illustration. Inview of the descriptions and illustrations, others skilled in the artcan, by applying, current knowledge, readily modify and/dr adapt thepresent invention for various applications without departing from thebasic concept, and therefore such modifications and/or adaptations areintended to be within the meaning and scope of the appended claims.

1. A functionalized nanocrystal comprising a plurality of polynucleotidestrands of predetermined sequence wherein a terminal portion of each ofthe plurality of polynucleotide strands is operably linked to thefunctionalized nanocrystal, and wherein the opposite terminus of each ofthe plurality polynucleotide strand is unbound to the functionalizednanocrystal and extends outwardly from the functionalized nanocrystal.2. The functionalized nanocrystal according to claim 1, wherein reactivefunctionalities are used to operably link the plurality ofpolynucleotide strands to the functionalized nanocrystal.
 3. Thefunctionalized nanocrystal according to claim 2, wherein thefunctionalized nanocrystal comprises reactive functionalities, andwherein a reactive functionality of the reactive functionalities isselected from the group consisting of an amino group, a carboxyl group,thiol-reactive group, and a combination thereof.
 4. The functionalizednanocrystal according to claim 2, wherein each polynucleotide strand ofthe plurality of polynucleotide strand comprises a reactivefunctionality selected from the group consisting of an amino-reactivegroup, a carboxyl-reactive group, and a thiol group.
 5. Thefunctionalized nanocrystal according to claim 1, wherein a linker isused to operably link a polynucleotide strand of the plurality ofpolynucleotide strands to the functionalized nanocrystal.
 6. Thefunctionalized nanocrystal according to claim 5, wherein thefunctionalized nanocrystal further comprises linker comprising avidin,wherein each polynucleotide strand of the plurality of polynucleotidestrands further comprises biotin, and wherein the biotin is bound to theavidin.
 7. The functionalized nanocrystal according to claim 1, whereinthe functionalized nanocrystal comprises a semiconductor nanocrystal. 8.The functionalized nanocrystal according to claim 1, wherein thefunctionalized nanocrystal comprises a doped metal oxide nanocrystal. 9.The functionalized nanocrystal according to claim 1, further comprisingmolecular probe operably linked to the functionalized nanocrystal. 10.The functionalized nanocrystal according to claim 9, wherein molecularprobe and the functionalized nanocrystal are operably linked using meansselected from the group consisting of by a reactive functionality on oneor more of the plurality of polynucleotide strands and a reactivefunctionality associated with the molecular probe, by a reactivefunctionality on the coating of the functionalized nanocrystal and areactive functionality associated with the molecular probe, by a linkerwhich has one portion that binds to a reactive functionality on one ormore polynucleotide strands and another portion which binds to areactive functionality associated with the molecular probe, by a linkerwhich has one portion that hybridizes to one or more polynucleotidestrands and another portion which hybridizes to the molecular probecomprising a nucleic acid molecule, and by synthesizing the molecularprobe as part of one or more polynucleotide strands.
 11. Thefunctionalized nanocrystal according to claim 9, wherein molecular probeand the functionalized nanocrystal are operably linked, wherein thefunctionalized nanocrystal further comprises avidin, wherein molecularprobe further comprises biotin, and wherein the biotin is bound to theavidin.
 12. A plurality of functionalized nanocrystals comprised of afunctionalized nanocrystal according to claim 1, wherein a firstspecies, primary dots, of the plurality of functionalized nanocrystalscomprises a plurality of polynucleotide strands of a predeterminedsequence; and a second species, secondary dots, of the plurality offunctionalized nanocrystals comprises a plurality of polynucleotidestrands of a sequence complementary to the sequence of the plurality ofpolynucleotide strands of the primary dot.
 13. A plurality offunctionalized nanocrystals comprised of a functionalized nanocrystalaccording to claim 9, wherein a first species, primary dots, of theplurality of functionalized nanocrystals comprises a plurality ofpolynucleotide strands of predetermined sequence; and a second species,secondary dots, of the plurality of functionalized nanocrystalscomprises a plurality of polynucleotide strands of a sequencecomplementary to the sequence of the plurality of polynucleotide strandsof the primary dot.
 14. The functionalized nanocrystal according toclaim 3, wherein the primary dots and secondary dots are of a uniformsize.
 15. The functionalized nanocrystals according to claim 12, whereinpolynucleotide strands of the plurality of polynucleotide strands of theprimary dots are hybridized to polynucleotide strands of the pluralityof polynucleotide strands of the secondary dots.
 16. The functionalizednanocrystals according to claim 13, wherein polynucleotide strands ofthe plurality of polynucleotide strands of the primary dots arehybridized to polynucleotide strands of the plurality of polynucleotidestrands of the secondary dots.